Recombinant expression of a putative prophage amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its biofilm

Authors: SIMMONS, MUSTAFA - US Department Of Agriculture (USDA); Morales, Cesar; Oakley, Brian; Seal, Bruce

Submitted to: Probiotics and Antimicrobial Proteins
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/11/2011
Publication Date: 10/11/2012
Citation: Simmons, M., Morales, C., Oakley, B., Seal, B.S. 2012. Recombinant expression of a putative prophage amidase cloned from the genome of Listeria monocytogenes that lyses the bacterium and its biofilm. Probiotics and Antimicrobial Proteins. 4:1-10.

Interpretive Summary: The bacterium Listeria monocytogenes is an important bacterial food-borne human pathogen which although rare may cause a sometimes fatal disease listeriosis. Listeria can be associated with uncooked meats including poultry, uncooked vegetables, soft cheeses and unpasteurized milk. The bacterium may be carried by animals without signs of diseases, can replicate at refrigerated temperatures and is frequently associated with biofilms. A biofilm is an aggregate of microorganisms in which cells adhere to each other and to an inanimate or biological surface. Biofilms are important to the survival and growth of bacteria in the food industry because Listeria growing in biofilms are protected against cleaning and disinfection, hence are very difficult to eradicate. A genomics analysis approach was used to identify bacteriophage lytic enzyme gene sequences in the DNA genomes of Listeria monocytogenes isolates. Subsequently, the gene for a bacteriophage lysin protein was cloned and expressed as a recombinant protein. The recombinant bacteriophage lysin was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot assays but was not active against other bacteria beyond the genus. Although the lysin protein alone reduced a listeria biofilm by only twenty percent, complete digestion of the biofilm was accomplished in conjunction with a protease. Consequently, the important food-borne bacterial pathogen Listeria monocytogenes can now possibly be controlled, even in biofilms, by use of bacteriophage lytic proteins.

Technical Abstract: Listeria monocytogenes is a Gram-positive, non-sporeforming, catalase-positive rod that is a major bacterial food-borne disease agent, causing listeriosis. Listeria can be associated with uncooked meats including poultry, uncooked vegetables, soft cheeses and unpasteurized milk. The bacterium can be carried by animals without signs of diseases, can replicate at refrigerated temperatures and is frequently associated with biofilms. Genomics analyses were used to identify lytic enzyme gene sequences in the genomes of Listeria monocytogenes isolates. PCR primers were designed that amplified nucleotide sequences of an N-acetylmuramoyl-L-alanine amidase gene from a L. monocytogenes strain 4b. The resultant amplification products were cloned into expression vectors, propagated in E. coli Rosetta strains and the recombinant protein was purified to homogeneity. Gene and protein sequencing revealed that the predicted and chemically determined amino acid sequence of the recombinant protein was homologous to N-acetylmuramoyl-L-alanine amidases. The recombinant lytic enzyme was capable of lysing both the parental L. monocytogenes strain as well as other strains of the bacterium in spot assays but was not active against other bacteria beyond the genus. A microtiter plate assay was utilized to assay for the ability of the recombinant amidase to digest a L. monocytogenes biofilm. Protease or lysozyme digestion did not significantly reduce the L. monocytogenes biofilm. Although the amidase alone reduced the biofilm by only twenty percent, complete digestion of the biofilm was accomplished in conjunction with a protease.